Research and development for renewable sources of biofuels has centered in large part on efficient means of converting abundant forms of biomass (cellulose, hemicellulose, and lignin of plant cell walls) into ethanol. However, because plant oils have twice the energy (caloric) content per kilogram compared with carbohydrates and proteins, increasing oil accumulation in vegetative tissues of plants could have a greater impact on the use of biomass to generate bioelectricity or to produce biodiesel fuel and nutritional feed. In addition, using the abundant vegetative biomass as the vehicle for oil accumulation would increase the oil storage capacity of plants and would have the potential to provide abundant supplies of plant oils for biodiesel production without diverting food production resources, thereby avoiding the competition between food and biofuel.
The most readily useful forms of plant oils are the diacyl- and, more particularly, triacylglycerol fatty acid compounds (DAGs and TAGs, respectively) in which two or three fatty acid chains are esterified to a glycerol backbone. Plant seeds constitute the normal repository for TAG compound accumulation. Because plant seed oils, particularly from oilseed crops, are mostly used for food or in some instances for a source of unusual, modified fatty acid compounds as chemical feedstocks, their use as biofuel is untenable.
However, as our understanding of the pathways of lipid/fatty acid synthesis, membrane formation and intracellular lipid trafficking has increased, so has the possibility of altering plants to increase the TAG content of the non-seed (vegetative) tissues. For a recent review of lipid transport (trafficking) see C. Benning (2009) Annual Review of Cell and Developmental Biology 25:71-91, the contents of which are incorporated herein by reference. If the abundant, vegetative tissues of plants were enabled to accumulate increased amounts of oils (TAG compounds) the harvestable energy, particularly from non-food crops, could be enormously increased. Ohlrogge and Chapman postulated that “Producing biomass with 10% oil on a dry weight basis could have a major positive impact on the recovery of energy from dedicated biomass crops” (the Biochemical Society, April 2011, pp 34-38). They further speculated that planting such a crop in place of the maize that is currently planted for ethanol production (˜12 million hectares) could result in 24 billion liters of biodiesel.
In developing seeds there are two pathways involved in TAG biosynthesis. The acyl-CoA-dependent pathway is catalyzed by diacylglycerol:acyl-CoA acyltransferase (DGAT) and the acyl-CoA-independent pathway by phospholipid:diacylglycerol acyltransferase (PDAT), which transfers an acyl group from phospholipids such as phosphatidylcholine (PC) to diacylglycerol. In oilseeds, TAGs are packaged in lipid droplets, which consist of a central core of TAG enclosed by a monolayer of phospholipids with a subset of specific proteins embedded therein. The most abundant proteins coating seed oil droplets are oleosins. The roles of oleosins are: 1) to stabilize lipid droplets during seed maturation and, 2) to protect TAG from hydrolysis by TAG lipases. Plant vegetative tissues normally do not express significant amounts of oleosins and they accumulate very limited amounts of storage lipids such as TAGs.
Various attempts to increase oil (TAG) storage in non-seed (vegetative) plant tissue have been described. Notable results have has been described in the works of Vanhercke, et al. (Plant Biotechnology Journal (2014) 12:231-239; and US Patent Application Publications US2013/0164798A1 and US2013/0247451A1). Alternative strategies are documented in Shanklin et al., US2014/0031573A1, which includes enlisting both acyl-CoA-dependent and acyl-CoA-independent TAG biosynthetic pathways to enhance TAG accumulation in non-seed tissue.
The approaches documented to date haven't yet produced a crop plant, neither biomass crop plant nor other crop plant, having stably heritable commercially significant improvements in the TAG accumulation in vegetative tissues. The present application provides a novel approach to enhancing oil (TAG) accumulation in the non-seed, vegetative tissues of plants.